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LAB. NO.

(2+3)

EXTRACTION AND

ISOLATION TECHNIQUES

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A) An Overview of Extraction Techniques for

Medicinal and Aromatic Plants

A wide range of technologies is available for the extraction of active components and essential

oils from medicinal and aromatic plants. The choice depends on the economic feasibility and

suitability of the process to the particular situation.

Extraction, as the term is used pharmaceutically, involves the separation of medicinally active

portions of plant or animal tissues from the inactive or inert components by using selective

solvents in standard extraction procedures.

General Methods of Extraction of Medicinal Plants:

1. Maceration

In this process, the whole or coarsely powdered crude drug is placed in a stoppered

container with the solvent and allowed to stand at room temperature for a period of at least 3

days with frequent agitation until the soluble matter has dissolved. The mixture then is strained,

the marc (the damp solid material) is pressed, and the combined liquids are clarified by filtration

or decantation after standing.

2. Infusion

Fresh infusions are prepared by macerating the crude drug for a short period of time with

cold or boiling water. These are dilute solutions of the readily soluble constituents of crude

drugs.

3. Digestion

This is a form of maceration in which gentle heat is used during the process of extraction.

It is used when moderately elevated temperature is not objectionable.

4. Decoction

In this process, the crude drug is boiled in a specified volume of water for a defined time;

it is then cooled and strained or filtered. This procedure is suitable for extracting water-soluble,

heat-stable constituents.

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5. Percolation

This is the procedure used most frequently to extract active ingredients in the preparation

of tinctures and fluid extracts. A percolator (a narrow, cone-shaped vessel open at both ends) is

generally used.

6. Hot Continuous Extraction (Soxhlet)

In this method, the finely ground crude drug is placed in a porous bag or “thimble” made

of strong filter paper, which is placed in chamber E of the Soxhlet apparatus. The extracting

solvent in flask A is heated, and its vapors condense in condenser D. The condensed extractant

drips into the thimble containing the crude drug, and extracts it by contact. When the level of

liquid in chamberErises to the top of siphon tube C, the liquid contents of chamber E siphon into

flask A. This process is continuous and is carried out until a drop of solvent from the siphon tube

does not leave residue when evaporated. The advantage of this method, compared to

previously described methods, is that large amounts of drug can be extracted with a much

smaller quantity of solvent.

7. Ultrasound Extraction (Sonication)

The procedure involves the use of ultrasound with frequencies ranging from 20 kHz to

2000 kHz; this increases the permeability of cell walls and produces cavitation.

8. Supercritical Fluid Extraction

Supercritical fluid extraction (SFE) is an alternative sample preparation method with general

goals of reduced use of organic solvents and increased sample throughput. The factors to

consider include temperature, pressure, sample volume, analyte collection, modifier (cosolvent)

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addition, flow and pressure control, and restrictors. Generally, cylindrical extraction vessels are

used for SFE and their performance is good beyond any doubt.

The collection of the extracted analyte following SFE is another important step:

significant analyte loss can occur during this step, leading the analyst to believe that the actual

efficiency was poor.

There are many advantages to the use of CO2 as the extracting fluid. In addition to its

favorable physical properties, carbon dioxide is inexpensive, safe and abundant. But while

carbon dioxide is the preferred fluid for SFE, it possesses several polarity limitations. Solvent

polarity is important when extracting polar solutes and when strong analyte-matrix interactions

are present. Organic solvents are frequently added to the carbon dioxide extracting fluid to

alleviate the polarity limitations. Of late, instead of carbon dioxide, argon is being used because

it is inexpensive and more inert.

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The component recovery rates generally increase with increasing pressure or temperature:

the highest recovery rates in case of argon are obtained at 500 atm and 150° C.

The extraction procedure possesses distinct advantages:

i) The extraction of constituents at low temperature, which strictly avoids damage

from heat and some organic solvents.

ii) No solvent residues.

iii) Environmentally friendly extraction procedure.

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Steps Involved in the Extraction of Medicinal Plants

In order to extract medicinal ingredients from plant material, the following sequential

steps are involved:

1) Size Reduction

The dried plant material is disintegrated by feeding it into a hammer mill or a disc

pulverized which has built-in sieves. The objective for powdering the plant material is to rupture

its organ, tissue and cell structures so that its medicinal ingredients are exposed to the extraction

solvent. Furthermore, size reduction maximizes the surface area, which in turn enhances the

mass transfer of active principle from plant material to the solvent.

2) Extraction

Extraction of the plant material is carried out in three ways:

- Cold aqueous percolation

- Hot aqueous extraction (decoction)

- Solvent extraction (cold or hot)

3) Filtration

The extract so obtained is separated out from the marc (exhausted plant material) by allowing

it to trickle into a holding tank through the built-in false bottom of the extractor, which is

covered with a filter cloth. The marc is retained at the false bottom, and the extract is received in

the holding tank. From the holding tank, the extract is pumped into a sparkler filter to remove

fine or colloidal particles from the extract.

4) Concentration

The enriched extract from percolators or extractors, known as miscella, is fed into a wiped

film evaporator where it is concentrated under vacuum to produce a thick concentrated extract.

The concentrated extract is further fed into a vacuum chamber dryer to produce a solid mass free

from solvent. Or the concentration could be done using:

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A) Rotary Evaporation which is a technique that employs a rotary evaporator (also called a

“rotavap”) in order to remove excess solvents from samples by applying heat to a rotating

vessel at a reduced pressure. An important concept that this technique applies is that

liquids boil when the vapor pressure is equal to the external pressure or atmospheric

pressure. The machine utilizes a lower pressure than atmospheric pressure which allows

solvents to boil at lower temperatures. Furthermore, the rotation increases the surface

area and therefore evaporation proceeds more rapidly.

B) Lyophilization:Freeze drying also known as lyophilization is an important process in

sample preparation and for the preservation and storage of biologicals, pharmaceuticals

and foods. Of the various methods of dehydration, freeze drying is especially suited for

substances that are heat sensitive. Other than food processing (e.g., coffee, whole

dinners), freeze drying has been extensively used in the development of pharmaceuticals

(e.g., antibiotics) and preservation of biologicals (e.g., proteins, plasma, viruses and cell

lines). Freeze drying is a process whereby water or other solvent is removed from frozen

material by converting the frozen water directly into vapor without the intermediate

formation of liquid water. The basis for this sublimation process involves the absorption

of heat by the frozen sample in order to vaporize the ice; the use of a vacuum pump to

enhance the removal of water vapor from the surface of the sample; the transfer of water

vapor to a collector; and the removal of heat by the collector in order to condense the

water vapor. In essence, the freeze dry process is a balance between the heat absorbed by

the sample to vaporize the ice and the heat removed from the collector to convert the

water vapor into ice.

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B) Isolation of Hesperidin from Orange Peel

Using Soxhlet Extractor

Introduction:

Hesperidin, a polyphenolic bioflavonoid, is the predominant flavonoid in orange peel and

other citrus fruits. The highest concentration of hesperidin can be found in the white parts and

pulps of the citrus peels. Hesperidin can also be found in green vegetables. Hesperidin is a

flavanone glycoside consisting of the flavone hesperitin bound to the disaccharide rutinose. The

sugar causes hesperidin to be more soluble than hesperitin.Figure below shows its structure.

Hesperidin is an antioxidant that enhances the action of vitamin C to lower cholesterol levels. It

is also known to have pharmacological action as an anti-inflammatory, antihistaminic, and

antiviral agent.

Hesperidin alone, or in combination with other citrus bioflavonoids (diosmin, for

example), is most often used for blood vessel conditions such as hemorrhoids, varicose veins,

and poor circulation (venous stasis). It is also used to treat lymphedema, a condition involving

fluid retention that can be a complication of breast cancer surgery. The molecular mechanism of

the inhibitory effect of hesperidin on bone resorption is not clear.

Hesperidin improves the health of capillaries by reducing the capillary permeability so

that it could treat leg ulcers caused by poor circulation, when used in combination with diosmin.

Hesperidin, in combination with diosmin and a compression dressing, seems to improve

healing of small ulcers (less than 10 cm) caused by poor blood circulation. Hesperidin is used to

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reduce hay fever and other allergic conditions by inhibiting the release of histamine from mast

cells. The possible anti-cancer activity of hesperidin could be explained by the inhibition of

polyamine synthesis.

The following doses have been studied in scientific research:

- For the treatment of hemorrhoids inside the anus: 150 mg of hesperidin plus 1350 mg of

diosmin twice daily for 4 days, followed by 100 mg of hesperidin and 900 mg of diosmin

twice daily for 3 days.

- For preventing the return of hemorrhoids inside the anus: 50 mg of hesperidin plus 450

mg of diosmin twice daily for 3 months.

- For the treatment of leg ulcers caused by poor blood circulation (venous stasis ulcers): a

combination of 100 mg of hesperidin and 900 mg of diosmin daily for up to 2 months.

Hesperidin can be isolated by two different methods:

A) The first method involves extracting the dried citrus peel successively with petroleum

ether followed by methanol. The petroleum ether removes the essential oils in the peel

and the methanol will extract the glycoside (hesperidin).

B) The second method uses an alkaline extraction of chopped orange peel and acidification

of the extract. The hesperidin can then be crystallized from the acidified extract. Because

of its highly insoluble, crystalline nature, hesperidin is one of the easiest flavonoids to

isolate.

Work Procedure:

A) The first method:

1) 150 mL petroleum ether (40 – 60°C) is filled in a 250 mL round bottom flask with

magnetic stir bar.

2) 50g dried and powdered orange peel are placed in the extraction sleeve of a Soxhlet

extractor and covered with a little glass wool.

3) A reflux condenser is put on the Soxhlet extraction unit, and then the reaction mixture is

stirred and heated for 4 hours under strong reflux.

4) The petroleum ether extract is discarded. In order to remove the adherent petroleum

ether, the content of the extraction sleeve is laid out in an extensive crystallization dish.

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5) Afterwards the substance is placed again in an extraction sleeve and, like before, but with

150mL methanol, extracted unless the solvent leaving the extraction sleeve is colourless

(1 to 2 hours).

6) The extract is evaporated at the rotary evaporator until syrup consistency is reached.

Theresidue is mixed with 50 mL of 6% acetic acid; the precipitated solid is the crude

hesperidine.

7) It is sucked off with a Buchner funnel, washed with 6% acetic acid and dried 60 °C until

it isconstant in weight.

8) Do chemical identification of hesperidin using ferric chloride test = the result will be

wine red color will be present.

9) For recrystallization, a 5% solution of the crude product in dimethyl sulfoxide is

producedunder stirring and heating to 60–80 °C. Afterwards the same amount of water is

added slowlywhilst stirring. When cooling to room temperature the hesperidine

precipitates. It is suckedoff, first washed with little warm water and then with iso-

propanol and dried in the desiccators until it is constant in weight.

B) The second method:

- The dried orange peels (50gm) were macerated with 200ml of aq. Alkaline solution (10%

NaOH) pH 8-9 in different alcoholic solutions (0, 30, 50, 70 % of methanol in water) for

few hours, After complete maceration the mixture was filtered by Buchner funnel. Then

the filtrate will be acidified with 10 % HCl. Then allow standing until formation of

hesperidin crystals.

Analytics

Reaction control with TLC

TLC-Conditions:

adsorbant: TLC plates GF254 normal silica

Mobile phasesystems: Ethyl acetate : Methanol : H2O (15:3:2)

Dichloromethane : Methanol (85:15)

Chloroform : methanol : H2O ( 23: 12: 2)

n-butanol : acetic acid : water (3 : 1 : 1)

Rf (hesperidin) = 0.2 ------ 0.8

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Extraction Scheme:

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Intruduction to TLC:

Chromatography is used to separate mixtures of substances into their components. All

forms of chromatography work on the same principle.

Thin-layer chromatography (TLC) is an extremely valuable analytical technique in the

lab. It provides a rapid separation of compounds, and thereby gives an indication of the number

and nature of the components of a mixture. The following are some common uses of Thin-Layer

Chromatography:

1. To determine the number of components in a mixture.

2. To determine the identity of two substances.

3. To monitor the progress of a reaction.

4. To determine the effectiveness of a purification (check purity).

5. To determine the appropriate conditions for a column chromatographic separation.

6. To monitor column chromatography.

Principles of TLC:

- TLC is normally done on a small glass or plastic plate coated with a thin layer of a solid

— the most common are silica (SiO2) or alumina (Al2O3). This is the stationary phase.

The mobile phase is an organic solvent or solvent mixture.

- The sample mixture is applied near the bottom of the plate as a small spot, then placed in

a jar containing a few ml of solvent. The solvent climbs up the plate by capillary action,

carrying the sample mixture along with it.

- Each compound in the mixture moves at a different rate, depending on its solubility in the

mobile phase and the strength of its absorption to the stationary phase. When the solvent

gets near the top of the plate, it is allowed to evaporate, leaving behind the components of

the mixture at various distances from the point of origin.

- The ratio of the distance a compound moves to the distance the solvent moves is the Rf

value (retention factor). This value is characteristic of the compound, the solvent, and

the stationary phase

- A pencil line is drawn near the bottom of the plate and a small drop of a solution of the

dye mixture is placed on it. Any labeling on the plate to show the original position of the

drop must also be in pencil. If any of this was done in ink, dyes from the ink would also

move as the chromatogram developed.

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- When the spot of mixture is dry, the plate is stood in a shallow layer of solvent in a

covered beaker. It is important that the solvent level is below the line with the spot on it.

- The reason for covering the beaker is to make sure that the atmosphere in the beaker is

saturated with solvent vapour. To help this, the beaker is often lined with some filter

paper soaked in solvent. Saturating the atmosphere in the beaker with vapour stops the

solvent from evaporating as it rises up the plate.

- As the solvent slowly travels up the plate, the different components of the dye mixture

travel at different rates and the mixture is separated into different coloured spots.

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What if the substances you are interested in are colorless?

There are two simple ways of getting around this problem (unless the chemical

compounds are colored):

1) Using fluorescence:

You may remember that I mentioned that the stationary phase on a thin layer plate often has a

substance added to it which will fluoresce when exposed to UV light. That means that if you

shine UV light on it, it will glow.

That glow is masked at the position where the spots are on the final chromatogram - even if those

spots are invisible to the eye. That means that if you shine UV light on the plate, it will all glow

apart from where the spots are. The spots show up as darker patches.

While the UV is still shining on the plate, you obviously have to mark the positions of the spots

by drawing a pencil circle around them. As soon as you switch off the UV source, the spots will

disappear again.

2) Showing the spots up chemically:

In some cases, it may be possible to make the spots visible by reacting them with

something which produces a coloured product. A good example of this is in

chromatograms produced from amino acid mixtures.The chromatogram is allowed to dry

and is then sprayed with a solution of ninhydrin. Ninhydrin reacts with amino acids to

give coloured compounds, mainly brown or purple.

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In another method, the chromatogram is again allowed to dry and then placed in an

enclosed container (such as another beaker covered with a watch glass) along with a

few iodine crystals.The iodine vapour in the container may either react with the spots on

the chromatogram, or simply stick more to the spots than to the rest of the plate. Either

way, the substances you are interested in may show up as brownish spots.

by spraying the dry chromatogram of mixture on the TLC with general indicator like

vanillin sulfuric acid (few crystals in 10% H2SO4 of methanol)

Or by applying ammonia vapor to the plate to produce visible colored spots.

Continued notes of TLC:

- In TLC, a plastic, glass or aluminum sheet is coated with a thin layer of silica gel (stationary

phase)

regular silica gel, followed by end-capping

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- A very small amount of a solution of the substance to be analyzed is applied in a small spot

with a capillary tube, ~1cm from the bottom of the TLC plate

- The TLC is developed in a chamber which contains the developing solvent (the mobile

phase).

- Once the solvent is within ~1-2 cm of the top of the TLC sheet, the TLC is removed from the

developing chamber and the farthest extent of the solvent (the solvent front) is marked with a

pencil.

- The solvent is allowed to evaporate from the TLC sheet in the hood.

- The spots are visualized using a UV lamp.

- The separated chemicals may be colorless, so several methods used to view the spots:

• Visualization of spots under a UV254 lamp. The adsorbent layer will thus fluorescence

light green by itself, but spots of analyte quench this fluorescence.

• Iodine vapors are a general unspecific color.

• Specific color reagents exist into which the TLC plate is dipped or which are sprayed on

the plate.

• Once visible, the Rf value of each spot can be determined:

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• The Rf is defined as the distance the center of the spot moved divided by the distance the

solvent front moved (both measured from the origin).

• Rfvalues can be used to aid in the identification of a substance by comparison to

standards.

• Two substances that have the same Rf value may be identical; those with different Rf

values are not identical.

Results and discussions: